Bridging Length Scales to Study Polymer Assembly: Utilizing Bayesian Analysis of FRET to Measure Microtubule Nucleation

Abstract

A variety of proteins can assemble into large polymers as an integral part of their biological function. Studying the biochemistry and biophysics of polymer formation often involves time resolvable measurements of the amount of polymer during assembly. Non-destuctive measurements of polymer can be divided into two categories: short (spectroscopy) and large (microscopy) length scale measurements. Microscopy measurements of polymer amount are often dependent on spatial non-uniformity of polymer, whereas spectroscopy measurements of polymer amount are often highly model dependent. Here we show how both large and small length scale measurements can be combined to validate the assumptions behind both measurements while incorporating both measurements to make more accurate measurements of polymer amount. We utilize this approach with FRET and fluorescence microscopy to measure the amount of tubulin in polymer (microtubules) in order to study microtubule nucleation in Xenopus egg extracts. In addition, this approach may be useful to study a wide variety of polymers, including actin filaments, microfilaments, and protein aggregates.